About 40,000 individuals annually require keratoplasty for corneal opacity in the USA and up to 10 million people world-wide suffer bilateral corneal blindness. Most affected individuals do not receive treatment because tissue is not available to transplant. Difficulty in obtaining donor corneas has spurred research into alternative approaches to corneal regeneration including bioengineering of tissues and direct cell-based therapy. The potential for cellular based therapy is facilitated by the fact that allogenic tissues are well tolerated and seldom rejected after transplantation. Recent advances in devices and in cell-based therapeutic approaches using animal models show great promise for bioengineering solutions to pathologies of the corneal stroma and endothelium. A major barrier to the advancement of cell-based technologies, however, is a source for cells. In the case of keratocytes of corneal stroma and corneal endothelial cells, as well as of adult stem cells, the potential for expansion in culture is limited, and the properties of the cells vary according to individual donors and preparation methods, presenting a challenge in terms of consistency and quality control. In this project we will develop a stem cell-based reagent for corneal engineering using human embryonic stem cells (hESC), taking advantage of two recent developments in hESC research. Our lab has demonstrated conversion of hESC into corneal progenitor cells. We showed that the corneal progenitor cells readily differentiate to human keratocytes. Simultaneously, other researchers described a new method to produce essentially limitless numbers of homogeneous neural crest cells from hESC. We propose to combine these techniques to generate a standardized hESC-derived stem cell reagent with the potential to differentiate directly into corneal keratocytes and corneal endothelial cells. The project will: 1) Define a standard operating procedure to generate corneal progenitor cells from hESC. (2) Demonstrate homogeneity, stability, safety, and cryo-storage of the hESC-derived corneal progenitor cells. (3) Develop two hESC-based fluorescent reporter cell lines used to assess the quality and homogeneity of the corneal keratocytes and endothelial cells derived from the hESC. (4) Demonstrate functionality of the hESC-derived corneal cells using in vitro functional assays. This project applies novel technical developments to address an important barrier to corneal cell- based regenerative medicine. Successful completion of this project will provide a well-defined, safe, cell-based reagent available in unlimited quantities for use in bioengineering and cytotherapeutic approaches for the amelioration of corneal blindness.